Over the past several years, new techniques in cryo-electron microscopy single particle analysis, a technique whereby images of identical protein complexes at many different angles are computationally combined to determine the 3D structure of the protein, have made it possible to achieve near-atomic resolution for protein complexes 500 kDa. Last year we published a ground-breaking study of the structure of beta-galacosidase at 3.2 Angstrom resolution. This year we determined the structure of a complex between E. coli beta-galactosidase and the cell-permeant inhibitor phenyl ethyl-beta D-thiogalactopyranoside (PETG), at 2.2 Angstrom resolution. Comparison of protein structures with and without bound PETG show the location of the ligand, and the changes in the active site of the enzyme with ligand binding. Some regions of the map, such as the periphery, are visualized at resolutions worse than 2.2 Angstroms, presumably due to intrinsic flexibility. Many other regions display features consistent with resolutions higher than 2.2 Angstrom, with clear visualization of the carbonyl backbone and delineation of side chain densities. Densities can also be identified for several water molecules, as well and magnesium and sodium ions near the active site. There is no reported crystal structure for the E. coli beta-galactosidase-PETG complex, but comparison with the structure of a PETG complex with the structurally unrelated Trichoderma reesei beta-galactosidase shows distinct differences both in ligand conformation and in active site architecture. The demonstration that single particle cryo-EM can be used to visualize details of ligand binding to metabolic enzyme complexes such as beta-galactosidase at resolutions of 2.2 Angstrom heralds a new era in the use of cryo-EM for drug discovery. In order to obtain higher resolution structures of glutamate receptors (mentioned in our membrane protein project), we developed a grid-coating technique that allowed us to control the position of the molecules on the grid. The grids were first coated in gold, then the gold coating was reacted with thiol bearing PEG groups, which causes the support to be both slightly thicker and highly hydrophilic. For the glutamate receptors, this coating allowed the receptors to move into the holes, rather than being attracted to the hydrophobic carbon support, allowing high resolution single particle analysis. In addition, during imaging, small amounts of particle movement due to interaction with the electron beam can significantly reduce the possible resolution of the final structure; this gold coating also reduced the movement of particles during illumination. The prospect that the determination of protein structures to atomic resolution will no longer be limited by size or by the need for crystallization represents a significant and exciting horizon in structural biology. Rather than simply using cryo-EM maps, typically in the 6 - 20 Angstrom resolution range, as an envelope in which to fit structures obtained by X-ray crystallography, there is the exciting prospect of using cryo-EM to derive de novo, high-resolution structural models of proteins in one or multiple functional conformational states. The stage is now set for the application of these methods to analyze structures of a wide variety of biologically and medically relevant multi-protein complexes and membrane protein assemblies, which have historically represented the most challenging frontier in structural biology.